Targeting ocular diseases with novel ape1/ref-1 inhibitors

ABSTRACT

Apurinic/apyrimidinic endonuclease 1 redox factor 1 (APE1/Ref-1) inhibitors for inhibiting ocular diseases are disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATION

The present disclosure is a continuation application of U.S. patentapplication Ser. No. 16/968,009, filed Aug. 6, 2020, which claimspriority to International Patent Application Serial No.PCT/US2019/017023 (published as WO 2019/157163), which was filed Feb. 7,2019, and which claims priority to U.S. Provisional Application No.62/628,093, filed Feb. 8, 2018, the disclosures of which are herebyincorporated by reference in their entireties.

BACKGROUND OF THE DISCLOSURE

The present disclosure relates generally to the use of3-[(5-(2,3-dimethoxy-6-methyl1,4-benzoquinoyl)]-2-nonyl-2-proprionicacid (APX3330) and/or its derivatives (e.g.,[(2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylidene]-N,N-diethylpentanamide](APX2009), and(2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronapthalen-2-yl)methylidene]-N-methoxypentanamide](APX2014)) for inhibiting ocular diseases.

Ocular neovascularization is the key pathobiological feature of diseaseslike proliferative diabetic retinopathy (PDR), retinopathy ofprematurity (ROP), and wet age-related macular degeneration (AMD), whichtogether are major causes of blindness (Campochiaro, 2013). In PDR andROP, abnormal blood vessels grow in and on the retina, while in wet AMD,neovessels grow from the pigmented, subretinal choroid layer into theretina. In all cases, neovessels disrupt retinal architecture and canhemorrhage, leading to blindness. Although the exact stimuli promotingneovascularization are not always well characterized, hypoxia andinflammation both play crucial roles. The currently used, FDA approvedpharmacological treatments for these diseases are all biologicstargeting the vascular endothelial growth factor (VEGF) signalingpathway, such as ranibizumab and aflibercept (Prasad et al., 2010).Although these therapeutic agents have been very successful, significantproportions of patients are resistant and refractory (Lux et al., 2007;Falavarjani and Nguyen, 2013). Moreover, serious side effects includinghemorrhage and endophthalmitis are possible. Therefore, development ofnovel therapeutic approaches targeting other signaling pathways iscrucial.

Inflammation and hypoxia pay crucial role in neovascularization.Treatments that impinge upon both proinflammatory and hypoxic signalingoffer a unique therapeutic strategy. One such potential target is thereduction-oxidation factor 1-apurinic/apyrimidinic endonuclease(Ref-1/APE1), an intracellular signaling nexus with important roles intransducing proangiogenic stimuli. This bifunctional protein has anendonuclease role essential for base excision repair (APE1), while theRef-1 activity is a redox-sensitive transcriptional activator (Shah etal., 2017). Ref-1 redox signaling is a highly regulated process thatreduces oxidized cysteine residues in specific transcription factors aspart of their transactivation (Xanthoudakis and Curran, 1992;Xanthoudakis et al., 1992; Evans et al., 2000; Lando et al., 2000; Nishiet al., 2002; Seo et al., 2002; Li et al., 2010; Fishel et al., 2011;Cardoso et al., 2012; Kelley et al., 2012; Luo et al., 2012; Zhang etal., 2013; Fishel et al., 2015; Logsdon et al., 2016). This redoxsignaling affects numerous transcription factors including HIF-1α,NF-κB, and others. The regulation of HIF-1α and NF-κB are particularlyrelevant to angiogenesis and eye diseases (Evans et al., 2000; Nishi etal., 2002; Seo et al., 2002; Fishel et al., 2011; Cardoso et al., 2012;Fishel et al., 2015; Logsdon et al., 2016).

Excitingly, Ref-1 activity can be targeted pharmacologically.3-[(5-(2,3-dimethoxy-6-methyl1,4-benzoquinoyl)]-2-nonyl-2-proprionicacid, (APX3330; formerly called E3330) is a specific Ref-1/APE1 redoxinhibitor. APX3330 has been extensively characterized as a direct,highly selective inhibitor of Ref-1 redox activity that does not affectthe protein's endonuclease activity (Luo et al., 2008; Fishel et al.,2010; Su et al., 2011; Cardoso et al., 2012; Luo et al., 2012; Zhang etal., 2013; Fishel et al., 2015). Ref-1/APE1 is highly expressed duringretinal development, and in retinal pigment epithelium (RPE) cells,pericytes, choroidal endothelial cells and retinal endothelial cells(Chiarini et al., 2000; Jiang et al., 2011; Li et al., 2014a), and moregenerally, Ref-1 is frequently upregulated in regions of tissues inwhich inflammation is present (Zou et al., 2009; Kelley et al., 2010).APX3330 was previously shown to block in vitro angiogenesis, asevidenced by proliferation, migration, and tube formation of retinal andchoroidal endothelial cells (Jiang et al., 2011; Li et al., 2014b).Indeed, APX3330 delivered intravitreally (directly into the eye) reducedneovascularization in the very low density lipoprotein receptor (VLDLR)knockout mouse model of retinal neovascularization (Jiang et al., 2011),and also in laser-induced choroidal neovascularization (L-CNV) (Li etal., 2014b), the most widely used animal model that recapitulatesfeatures of wet AMD (Grossniklaus et al., 2010).

While the lead clinical candidate is efficacious in preclinical cancerstudies, a second generation Ref-1 inhibitors that would have increasedefficacy in antiangiogenic and anti-inflammatory transcription factor(NF-κB, HIF-1α) inhibition, as well as new chemical properties, isdesired.

BRIEF DESCRIPTION

The present disclosure is directed to the use of3-[(5-(2,3-dimethoxy-6-methyl1,4-benzoquinoyl)]-2-nonyl-2-proprionicacid (APX3330) and/or its derivatives, such[(2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylidene]-N,N-diethylpentanamide](APX2009) and(2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronapthalen-2-yl)methylidene]-N-methoxypentanamide](APX2014), for inhibiting ocular neovascularization. Particularly, itwas found that APX2009 and APX2014 provided enhanced inhibition of Ref-1function in a DNA-binding assay compared to APX3330. Both compounds wereantiproliferative against human retinal microvascular endothelial cells(HRECs; GI50 APX2009: 1.1 μM, APX2014: 110 nM) and macaque choroidalendothelial cells (Rf/6a GI50 APX2009: 26 μM, APX2014: 5.0 μM). Bothcompounds significantly reduced the ability of HRECs and Rf/6a cells toform tubes at mid nanomolar concentrations compared to control, and bothsignificantly inhibited HREC and Rf/6a cell migration in a scratch woundassay.

Ex vivo, both APX2009 and APX2014 inhibited choroidal sprouting at lowmicromolar and high nanomolar concentrations respectively. In thelaser-induced choroidal neovascularization mouse model, intraperitonealAPX2009 treatment significantly decreased lesion volume by 4-foldcompared to vehicle (p<0.0001, ANOVA with Dunnett's post hoc tests),without obvious intraocular or systemic toxicity. Thus, Ref-1 inhibitionwith APX2009 and APX2014 blocks ocular angiogenesis in vitro and exvivo, and APX2009 is an effective systemic therapy for CNV in vivo,establishing Ref-1 inhibition as a promising therapeutic approach forocular neovascularization.

Accordingly, in one aspect, the present disclosure is directed to amethod of inhibiting ocular neovascularization in a subject in needthereof. The method includes administering to the subject an effectiveamount of an apurinic/apyrimidinic endonuclease 1 redox factor 1(APE1/Ref-1) inhibitor, pharmaceutically acceptable salts orpharmaceutically acceptable solvates thereof.

In another aspect, the present disclosure is directed to a method ofinhibiting ocular neovascularization in a subject in need thereof, themethod comprising administering to the subject an effective amount of anapurinic/apyrimidinic endonuclease 1 redox factor 1 (APE1/Ref-1)inhibitor, pharmaceutically acceptable salts or pharmaceuticallyacceptable solvates thereof.

In another aspect, the present disclosure is directed to a method oftreating retinopathy of prematurity (ROP) in a subject in need thereof,the method comprising administering to the subject an effective amountof an apurinic/apyrimidinic endonuclease 1 redox factor 1 (APE1/Ref-1)inhibitor, pharmaceutically acceptable salts or pharmaceuticallyacceptable solvates thereof.

In yet another aspect, the present disclosure is directed to a method oftreating wet age-related macular degeneration (AMD) in a subject in needthereof, the method comprising administering to the subject an effectiveamount of an apurinic/apyrimidinic endonuclease 1 redox factor 1(APE1/Ref-1) inhibitor, pharmaceutically acceptable salts orpharmaceutically acceptable solvates thereof.

DESCRIPTION OF THE FIGURES

The disclosure will be better understood, and features, aspects andadvantages other than those set forth above will become apparent whenconsideration is given to the following detailed description thereof.Such detailed description makes reference to the following drawings,wherein:

FIGS. 1A & 1B depict the synthesis and activity of Ref-1 inhibitors.FIG. 1A depicts the synthetic scheme for APX2009 (6a) and APX2014 (6b).Structure of APX3330 (7) included for reference. Reagents andconditions: a, 2-iodo-3-hydroxy-1,4-naphthoquinone (iodolawsone, 1),2-propylacrylic acid (2), K₂CO₃, Pd(OAc)₂, argon, 100° C., 1 hour, 74%;b, (COCI)₂, DMF, DCM, RT overnight, 100%; c, DEA HCl (APX2009) orCH₃ONH₂·HCl (APX2014), DIPA HCl, RT, 45 minute, 62% and 71%respectively; d, NaOCH₃/CH₃OH, argon, 30 minutes, RT, 96% and 86%,respectively. FIG. 1B shows that APX2009 and APX2014 are more effectiveinhibitors of Ref-1-induced AP-1 DNA binding than APX3330 in an EMSA.Two separate gels from the same experiment are shown. The IC50 for redoxEMSA inhibition was 25, 0.45 and 0.2 μM for APX3330, APX2009 andAPX2014, respectively. These assays were performed multiple times withsimilar results.

FIGS. 2A-2D depict compounds APX2009 and APX2014 inhibit endothelialcell proliferation in HRECs and Rf/6a cells in vitro. Dose dependenteffects of APX2009 (FIG. 2A) and APX2014 (FIG. 2B) in human retinalendothelial cells (HRECs), and dose dependent effects of APX2009 (FIG.2C) and APX2014 (FIG. 2D) in Rf/6a choroidal endothelial cells. In vitroproliferation was measured using an alamarBlue assay. Median growthinhibition (GI₅₀) values are indicated. Mean±S.E.M., n=3 per dose.

FIGS. 3A-3D depict compounds APX2009 and APX2014 inhibit S phase inHRECs. After treating HRECs with the indicated concentrations of APX2009and APX2014, (FIG. 3A) EdU (red) and (FIG. 3C) Ki-67 (green) weredetected and nuclei (blue) stained with DAPI; Scale bar=100 μm. FIG. 3Bshows quantification of EdU and FIG. 3D shows quantification of Ki-67 inHRECs. Mean±S.E.M., n=3 fields per dose. **, p<0.01; ****, p<0.0001compared to DMSO control (one-way ANOVA with Dunnett's post hoc test).Representative data from three independent experiments. See FIGS. 4A & 5.

FIG. 4A depicts full fields of the EdU staining for all doses (sameexperiment as FIGS. 3A-3D) show that APX2009 and APX2014 decreased DNAsynthesis dose dependently in HRECs. Scale bars=100 μm.

FIG. 4B depicts propidium iodide cell cycle profiles for indicatedtreatments.

FIG. 4C shows quantification of cell cycle phase. Mean±S.E.M., n=3independent experiments.

FIG. 5 depicts separate channel images of Ki-67 staining for all doses(same experiment as FIGS. 3A-3D) show that APX2009 and APX2014 decreasedproliferation dose dependently in HRECs. Scale bars=100 μm.

FIGS. 6A & 6B depict that APX2009 and APX2014 did not induce cell deathin HRECs. FIG. 6A shows TUNEL staining (red) for cell death and DAPI(blue) for nuclear staining. No TUNEL-positive cells are observed inthese images. Staurosporine acts as a positive control. Scale bar=100μm. FIG. 6B shows quantification data showing the percentage of TUNELpositive cells upon various treatments. Mean±S.E.M., n=3. ns,non-significant (One-way ANOVA with Dunnett's post hoc tests).Representative data from two independent experiments.

FIGS. 7A-7D show that compounds APX2009 and APX2014 inhibitedendothelial cell migration in HRECs and Rf/6a cells in vitro. FIG. 7Adepicts the effect of APX2009 and APX2014 on cell migration in HRECs. Aconfluent monolayer of HRECs with various treatments (highest dosesshown) was wounded and wound closure was monitored for 8 hours. FIG. 7Bshows quantitative analysis of cell migration, showing that APXcompounds significantly block the migration of HRECs. FIG. 7C depictsthe effects of APX2009 and APX2014 on cell migration in Rf/6a cells. Aconfluent monolayer of Rf/6a with various treatments (highest dosesshown) was wounded and wound closure was monitored for 16 hours.

FIG. 7D shows quantitative analysis of cell migration, showing that APXcompounds significantly block the migration of Rf/6a cells. Mean±S.E.M.,n=3 per dose. **, p<0.01; ***, p<0.001 compared to DMSO control (one-wayANOVA with Dunnett's post hoc test). Scale bar=500 μm.

FIGS. 8A-8D depict APX2009 and APX2014 inhibited migration of HRECs andRf/6a cells in vitro. The effect of (FIG. 8A) APX2009 and (FIG. 8B)APX2014 on cell migration in HRECs is shown. A confluent monolayer ofHRECs treated with various concentrations of each compound was woundedand wound closure was monitored for 8 hours. The effect of (FIG. 8C)APX2009 and (FIG. 8D) APX2014 on cell migration in Rf/6a cells is shown.A confluent monolayer of Rf/6a cells treated with various concentrationsof each compound was wounded and wound closure was monitored for 16hours. Scale bars=500 μm.

FIGS. 9A-9D depict compounds APX2009 and APX2014 inhibited endothelialtube formation in HRECs and Rf/6a cells in vitro. FIG. 9A depicts tubeformation on Matrigel by HRECs in the presence of the indicatedconcentrations of APX compounds; FIG. 9B shows a quantitative analysisof APX2009 and APX2014 compounds on HREC tube formation. Tubular lengthwas measured and represented as relative to DMSO control. FIG. 9Cdepicts tube formation on Matrigel by Rf/6a in the presence of theindicated concentrations of APX compounds; FIG. 9D shows a quantitativeanalysis of APX2009 and APX2014 compounds on Rf/6a tube formation.Tubular length was measured and represented as relative to DMSO control.Mean±S.E.M., n=3 wells. **, p<0.01; ***, p<0.001 compared to DMSOcontrol (one-way ANOVA with Dunnett's post hoc test). Representativedata from three independent experiments. Scale bar=500 μm.

FIGS. 10A-10D depict APX2009 and APX2014 inhibited endothelial tubeformation in HRECs in vitro. Tube formation on Matrigel by HRECs in thepresence of the indicated concentrations of APX2009 (FIG. 10A) and theindicated concentrations of APX2014 (FIG. 10B) is shown. Further, tubeformation on Matrigel by Rf/6a cells in the presence of the indicatedconcentrations of APX2009 (FIG. 10C) and in the presence of theindicated concentrations of APX2014 (FIG. 10D) is shown. Scale bars=500μm.

FIGS. 11A-11D depict compounds APX2009 and APX2014 inhibit TNF-αmediated NF-κB signaling and proangiogenic target gene mRNA expression.After treating HRECs with the indicated concentrations of APX2009 andAPX2014, p65 (red) was detected by immunofluorescence and nuclei (blue)stained with DAPI; compounds dose-dependently reduced p65 nucleartranslocation as evidenced by decreased overlap between red and bluesignals. BAY 11-7082 is a positive control NF-κB inhibitor. Scalebar=100 μm. (FIG. 11B) VEGFA, (FIG. 11C) VCAM1, and (FIG. 11D) CCL20mRNA expression levels in HRECs. APX2009 and APX2014 dose dependentlyinhibited levels of each transcript. Mean±S.E.M., n=3 technicalreplicates. *, p<0.05; **, p<0.01; ***, p<0.001 compared to DMSO control(one-way ANOVA with Dunnett's post hoc test). Representative data fromthree independent experiments.

FIGS. 12A-12D depict compounds APX2009 and APX2014 inhibited choroidalsprouting in a concentration-dependent manner. FIG. 12A isrepresentative phase contrast images of choroidal sprouts formed 48hours after treatment with indicated APX2009 concentrations or vehicle(0.5% DMSO) control. FIG. 12B shows quantification of sprouting distancefrom the edge of the APX2009-treated choroidal tissue piece to the endof the sprouts averaged from four perpendicular directions using ImageJsoftware. FIG. 12C is representative images of choroidal sprouts formed48 hours after treatment with indicated APX2014 concentrations orvehicle (0.2% DMSO) control. FIG. 12D shows quantification of sproutingdistance from the edge of the choroidal tissue piece to the end of thesprouts averaged from four perpendicular directions using ImageJsoftware. Mean±S.E.M., n=4-5 choroids/per treatment; N=3-4 eyes. ***,p<0.001; ****, p<0.0001 (ANOVA with Dunnett's post hoc test). Scalebars=500 μm.

FIGS. 13A-13C depict systemic Ref-1 inhibition with APX3330 blockedneovascularization in the laser-induced choroidal neovascularization(L-CNV) mouse model. FIG. 13A shows representative optical coherencetomography (OCT) images obtained 7 days post-laser, showing CNV lesionsin eyes of vehicle (left) and 50 mg/kg i.p. APX3330 (right) treatedanimals. FIG. 13B shows representative images from confocal microscopyfor agglutinin-stained CNV lesions 14 days post-laser treatment. FIG.13C shows quantification of CNV lesion vascular volumes from Z-stackimages at day 14 using ImageJ software. Mean±S.E.M., n=7-9eyes/treatment. * p<0.05 (unpaired Student's t-test). Scale bars=100 μm.

FIGS. 14A-14D depict intraperitoneal APX2009 inhibited choroidalneovascularization in the L-CNV mouse model. FIG. 14A showsrepresentative OCT images obtained 7 and 14 days post-laser, showing CNVlesions of untouched control, vehicle, 12.5 mg/kg and 25 mg/kg APX2009compound i.p. injected twice daily until 14 days post-laser treatment.FIG. 14B depicts fluorescein angiography (FA) of CNV showing thevascular leakage suppression by APX2009. FIG. 14C is representativeimages from confocal microscopy for agglutinin-stained CNV lesions 14days post-laser treatment. FIG. 14D shows quantification of CNV lesionvascular volumes from Z-stack images at day 14 using ImageJ software.Mean±S.E.M., n=8-10 eyes/treatment. ns, non-significant; ***, p<0.001compared to DMSO control (one-way ANOVA with Tukey's post hoc test).Scale bars=100 μm.

FIGS. 15A-15C depict APX2009 inhibited choroidal neovascularization inthe L-CNV mouse model. FIG. 15A depicts Double-stained Agglutinin andGriffonia simplicifolia isolectin B4 (GSIB4) confocal images in theL-CNV lesions 14 days post-laser treatment. FIG. 15B showsquantification of CNV lesion vascular volumes from Z-stack ofGS-IB4-stained images at day 14 using ImageJ software. ns,non-significant; ****, p<0.0001 (One-way ANOVA with Tukey's post hoctests). FIG. 15C shows quantification of mouse body weight of vehicleand APX2009 injected groups over 14 days. No significant difference inweight between treatments was observed at any time point (repeatedmeasures two-way ANOVA). Mean S.E.M., n=8-10 eyes/treatment. Scalebars=100 μm.

FIG. 16 depicts sample data of known HIF-regulated genes that aredownregulated by indicated concentrations of APX2009 in HRECs. RNA-Seqdata were analyzed by principal component (PC) regression, withsignificant genes having r>0.42 with respect to association with the PCof APX2009 treatment. Pathway enrichment analysis revealed enrichment ofthese genes regulated by HIF1A (p=0.02).

FIG. 17 depicts Ref-1 being upregulated in wet AMD. Sections of humaneye stained for Ref-1 (brown) revealed expression in nuclei of the innernuclear layer (INL), outer nuclear layer (ONL), and choroid,specifically in wet AMD, but not age-matched control. Scale bar=50 μm.GCL, ganglion cell layer.

DETAILED DESCRIPTION

The present disclosure relates generally to APE1 inhibitors, such as3-[(5-(2,3-dimethoxy-6-methyl1,4-benzoquinoyl)]-2-nonyl-2-proprionicacid (APX3330) and/or its derivatives (e.g.,[(2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylidene]-N,N-diethylpentanamide](APX2009) and(2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronapthalen-2-yl)methylidene]-N-methoxypentanamide](APX2014)) for inhibiting ocular neovascularization. Moreover, thepresent disclosure is directed to the use of APX2009 and APX2014 fortreating diseases like proliferative diabetic retinopathy (PDR),retinopathy of prematurity (ROP), and wet age-related maculardegeneration (AMD).

In suitable embodiments, the present disclosure includes administeringto a subject in need thereof an effective amount of an APE1 inhibitor,the APE1 inhibitor capable of interacting with the APE1 protein such tocause unfolding of the APE1 protein in the amino terminal portion ofAPE1, inhibiting the ability of APE1 to interact with other proteins inthe neurons or to perform its redox signaling function. Moreparticularly, APE1 inhibitors used in the present disclosure block theability of APE1/Ref-1 to convert NF-κB and AP-1 from an oxidised stateto reduced state, thereby altering their transcriptional activity.

Accordingly, in particular suitable embodiments, the APE1 inhibitor hasthe formula:

wherein R₁ is selected from the group consisting of alkyl, alkoxy,hydroxyl, and hydrogen; R₃ and R₆ are independently selected from thegroup consisting of a substituted or unsubstituted alkoxy, a substitutedor unsubstituted aryl and an oxo; R₄ and R₅ are independently selectedfrom the group consisting of an alkoxy and aryl, or both R₄ and R₅ takentogether form a substituted or unsubstituted napthoquinone;

-   -   X is selected from the group consisting of CH═CR₂ and NCH,        wherein R₂ is selected from the group consisting of C₁-C₁₀ alkyl        and CF₃CH₂CH₂; and    -   Y is selected from the group consisting of N(Rz)R2 or        NR{circumflex over ( )}OR{circumflex over ( )}, wherein each Rz        is independently selected from the group consisting of C₁-C₆        alkyl, heteroalkyl, cycloalkyl and cycloheteroalkyl, straight or        branched chain or optionally substituted, or both Rz and R2        taken together with the attached nitrogen form an optionally        substituted heterocycle; where each R{circumflex over ( )} is        independently selected from the group consisting of hydrogen,        alkyl, heteroalkyl, cyclohexyl, and cycloheteroalkyl, each of        which is optionally substituted, or both R{circumflex over ( )}        are taken together with the attached nitrogen and oxygen to form        an optionally substituted heterocycle.

Particularly suitable APE1 inhibitors include3-[(5-(2,3-dimethoxy-6-methyl1,4-benzoquinoyl)]-2-nonyl-2-proprionicacid, (hereinafter “E3330” or “3330” or “APX3330”), and/or its analogs(e.g.,[(2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylidene]-N,N-diethylpentanamide](hereinafter “APX2009”),(2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronapthalen-2-yl)methylidene]-N,N-dimethylpentanamide](hereinafter “APX2007”),(2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronapthalen-2-yl)methylidene]-N-methoxypentanamide](hereinafter “APX2014”),(2E)-2-(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-N,N,2-trimethylprop-2-enamide(hereinafter “APX2032”)). Additional suitable analogs are shown belowand in Table 1. Further information on APX3330 may be found in Abe etal., U.S. Pat. No. 5,210,239, and information on APX2009 may be found inKelley et al., J Pharmacol Exp Ther. 2016 November, 359(2): 300-309,each incorporated herein by reference to the extent they are consistentherewith.

TABLE 1 COMPOUND ID R₁ X C(═O)Y R₂ R₃ R₄ R₅ R₆ EF MW APX3330 CH₃ CH═CR₂OH C₂H₁₉ ═O MeO MeO ═O C₂₁H₃₀O₆ 378.459 APX2006 MeO CH═CR₂ NMe C₃H₇ ═Onapthoquinone ═O C₁₈H₁₉NO₄ 313.353 APX2007 MeO CH═CR₂ N(Me)₂ C₃H₇ ═Onapthoquinone ═O C₁₉H₂₁NO₄ 327.38 APX2008 MeO CH═CR₂ NEt C₃H₇ ═Onapthoquinone ═O C₁₉H₂₁NO₄ 327.38 APX2009 MeO CH═CR₂ N(Et)₂ C₃H₇ ═Onapthoquinone ═O C₂₁H₂₅NO₄ 355.428 APX2010 CH3 CH═CR₂ NCH₃ C₄H₉ ═Onapthoquinone ═O C₁₇H₂₃NO₅ 321.373 APX2011 CH3 CH═CR₂ N(CH₃)₂ C₄H₉ ═Onapthoquinone ═O C₂₀H₂₃NO₃ 325.408 APX2012 CH₃ CH═CR₂ NCH₂CH₃ C₄H₉ ═Onapthoquinone ═O C₂₀H₂₃NO₃ 325.408 APX2013 CH₃ CH═CR₂ N(Et)₂ C₄H₉ ═Onapthoquinone ═O C₂₂H₂₇NO₃ 353.462 APX2014 MeO CH═CR₂ NOMe C₃H₇ ═Onapthoquinone ═O C₁₈H₁₉NO₅ 329.352 APX2015 CH₃ CH═CR₂ N-cPro C₄H₉ ═Onapthoquinone ═O C₂₁H₂₃NO₃ 337.419 APX2016 CH₃ CH═CR₂ NOMe C₄H₉ ═Onapthoquinone ═O C₁₉H₂₁NO₄ 327.38 APX2017 CH₃ CH═CR₂ N-Et-Pip C₄H₉ ═Onapthoquinone ═O C₂₄H₃₀N₂O₃ 394.515 APX2018 CH₃ CH═CR₂ N-cHexyl C₄H₉ ═Onapthoquinone ═O C₂₄H₂₉NO₃ 379.492 APX2019 CH₃ CH═CR₂ 2-Piperdone C₄H₉═O napthoquinone ═O C₂₂H₂₄N₂O₄ 380.444 APX2020 CH₃ CH═CR₂ N(Me)OMe C₄H₉═O napthoquinone ═O C₂₀H₂₃NO₄ 341.407 APX2021 CH₃ CH═CR₂ E-MorpholinoC₄H₉ ═O napthoquinone ═O C₂₂H₂₅NO₄ 367.445 APX2022 CH₃ CH═CR₂Z-Morpholino C₄H₉ ═O napthoquinone ═O C₂₂H₂₅NO₄ 367.445 APX2023 CH₃CH═CR₂ NH2 C₄H₉ ═O napthoquinone ═O C₁₈H₁₉NO₃ 297.348 APX2024 CH₃ CH═CR₂E-NCH₂CH₂OMe C₄H₉ ═O napthoquinone ═O C₂₁H₂₅NO₄ 355.434 APX2025 CH₃CH═CR₂ Z-NCH₂CH₂OMe C₄H₉ ═O napthoquinone ═O C₂₁H₂₅NO₄ 355.434 APX2026Cl CH═CR₂ NOMe C₃H₇ ═O napthoquinone ═O C₁₇H₁₆ClNO₄ 333.77 APX2027 ClCH═CR₂ N(Et)₂ C₃H₇ ═O napthoquinone ═O C₂₀H₂₂ClNO₃ 359.85 APX2028 OHCH═CR2 OH C3H7 ═O napthoquinone ═O C16H14O5 286.283 APX2029 MeO CH═CR₂N(Et)₂ C₃H₇ ═O napthoquinone ═O C₂₁H₂₅NO₄ 355.434 APX2030 Me CH═CR₂N(Me)₂ C₃H₇ ═O napthoquinone ═O C₁₉H₂₁NO₃ 311.381 APX2031 MeO CH═CR₂NCH₃ CH₃ ═O napthoquinone ═O C₁₆H₁₅NO₄ 285.295 APX2032 MeO CH═CR₂N(CH₃)₂ CH₃ ═O napthoquinone ═O C₁₇H₁₇NO₄ 299.321 APX2033 MeO CH═CR₂ OHCH₃ ═O napthoquinone ═O C₁₅H₁₂O₅ 272.253 APX2034 MeO CH═CR₂ OH C₃H₇ ═Onapthoquinone ═O C₁₇H₁₆O₅ 300.306 APX2043 MeO CH═CR₂ N(CH₃)₂ C₃H₇ OHnapthoquinone OH C₁₉H₂₅NO₄ 331.412 APX2044 CF₃O CH═CR₂ N(Et)₂ C₃H₇ ═Onapthoquinone ═O C₂₁H₂₂F₃NO₄ 409.405 APX2045 CH₃ CH═CR₂ N(Et)₂ C₃H₇ ═Onapthoquinone ═O C₂₁H₂₅NO₃ 339.435 APX2046 CH₃ CH═CR₂ N(Et)₂ CF₃CH₂CH₂═O napthoquinone ═O C₂₁H₂₂F₃NO₃ 393.406 APX2047 CH₃ CH═CR₂ N(Et)₂ C₃H₇OCH₃ napthoquinone OCH₃ C₂₃H₃₁NO₃ 369.505 APX2048 CH₃ CH═CR₂ NOCH₃ C₉H₁₉═O MeO MeO ═O C₂₃H₃₁NO₄ 397.515 APX2049 CH₃ CH═CR₂N(CH₃)CC(O)C(O)C(O)C(O)COH C₉H₁₉ ═O MeO MeO ═O C₂₈H₄₅NO₁₀ 555.665APX2050 CH₃ CH═CR₂ N(CH₃)OCH₃ C₉H₁₉ ═O MeO MeO ═O C₂₃H₃₅NO₆ 421.534

It has herein been found that the administration of APE1 inhibitors, andin particular, APX2009 and/or APX2014, inhibits APE1 protein frominteracting with other proteins in the neurons. Particularly, APX2009and APX2014 exert their antiangiogenic effects by blocking theactivation of transcription factors induced by Ref-1, likely candidatesincluding NF-κB and HIF-1α, both of which can regulate VEGF.

Suitable dosages of the APE1 inhibitor, pharmaceutically acceptablesalts or pharmaceutically acceptable solvates thereof, for use in themethods of the present disclosure will depend upon a number of factorsincluding, for example, age and weight of an individual, severity ofocular neovascularization-related disorder or disease to be treated,nature of a composition, route of administration and combinationsthereof. Ultimately, a suitable dosage can be readily determined by oneskilled in the art such as, for example, a physician, a veterinarian, ascientist, and other medical and research professionals. For example,one skilled in the art can begin with a low dosage that can be increaseduntil reaching the desired treatment outcome or result. Alternatively,one skilled in the art can begin with a high dosage that can bedecreased until reaching a minimum dosage needed to achieve the desiredtreatment outcome or result.

In one particularly suitable embodiment, the APE1/Ref-1 inhibitor isAPX2009, and the subject is administered from about 12.5 mg/kg to about35 mg/kg APX2009 per day.

In one particularly suitable embodiment, the APE1/Ref-1 inhibitor isAPX2014, and the subject is administered from about 12.5 mg/kg to about35 mg/kg APX2014 per day.

In some embodiments, the APE1 inhibitor is administered via acomposition that includes the APE1 inhibitor and a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers may be, forexample, excipients, vehicles, diluents, and combinations thereof. Forexample, where the compositions are to be administered orally, they maybe formulated as tablets, capsules, granules, powders, or syrups; or forparenteral administration, they may be formulated as injections(intramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, intravenous, intravitreal), drop infusionpreparations, or suppositories. These compositions can be prepared byconventional means, and, if desired, the active compound (e.g., APX2009,APX2014) may be mixed with any conventional additive, such as anexcipient, a binder, a disintegrating agent, a lubricant, a corrigent, asolubilizing agent, a suspension aid, an emulsifying agent, a coatingagent, or combinations thereof.

It should be understood that the pharmaceutical compositions of thepresent disclosure can further include additional known therapeuticagents, drugs, modifications of the synthetic compounds into prodrugs,and the like for alleviating, mediating, preventing, and treating thediseases, disorders, and conditions described herein. For example, inone embodiment, the APE1 inhibitor can be administered with one or moreof current therapeutic agents and drugs for treating ocularneovascularization (e.g., anti-VEGF therapies, including, for example,anti-VEGF biologics such as ranibizumab, bevacizumab, aflibercept;antisense RNA, RNA silencing or RNA interference (RNAi) of angiogenicfactors, including ribozymes that target VEGF expression; inhibitors ofthe SRPK family of kinases, FOVISTA® and other agents targeting plateletderived growth factor (PDGF); squalamine((1S,2S,5S,7R,9R,10R,11S,14R,15R)—N-{3-[(4-aminobutyl)amino]propyl}-9-hydroxy-2,15-dimethyl-14-[(2R,5R)-6-methyl-5-(sulfooxy)heptan-2-yl]tetracyclo[8.7.0.0{circumflexover ( )}{2,7}0.0{circumflex over ( )}{11,15}]heptadecan-5-aminium);X-82 (Tyrogenix, Needham Heights, Massachusetts); PAN-90806 (PanOptica,Bernardsville, New Jersey); TNP470 (Sigma-Aldrich, St. Louis, Missouri)and fumagillin(2E,4E,6E,8E)-10-{[(3R,4S,5S,6R)-5-methoxy-4-[(2R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl]oxy}-10-oxodeca-2,4,6,8-tetraenoicacid); protein kinase C inhibitors; inhibitors of VEGF receptor kinase;pigment epithelium derived factor (PEDF); endostatin; angiostatin;anecortave acetate; triamcinolone((11β,16α)-9-Fluoro-11,16,17,21-tetrahydroxypregna-1,4-diene-3,20-dione);verteporfin(3-[(23S,24R)-14-ethenyl-5-(3-methoxy-3-oxopropyl)-22,23-bis(methoxycarbonyl)-4,10,15,24-tetramethyl-25,26,27,28-tetraazahexacyclo[16.6.1.13,6.18,11.113,16.019,24]octacosa-1,3,5,7,9,11(27),12,14,16,18(25),19,21-dodecaen-9-yl]propanoicacid), porfimer sodium (photofrin)), vitamins and minerals (vitamins Cand E, beta-carotene, zinc, copper, lutein, zeaxanthin, omega-3 fattyacids), and the like).

The pharmaceutical compositions including the APE1 inhibitor and/orpharmaceutical carriers used in the methods of the present disclosurecan be administered to a subset of individuals/subjects in need. As usedherein, a “subject in need” refers to an individual at risk for orhaving an ocular disease and/or ocular neovascularization, or anindividual at risk for or having an ocular disease and/or a disease ordisorder associated with ocular neovascularization (e.g., retinopathy ofprematurity (ROP), proliferative diabetic retinopathy (PDR), diabeticretinopathy, wet age-related macular degeneration (AMD), pathologicalmyopia, hypertensive retinopathy, occlusive vasculitis, polypoidalchoroidal vasculopathy, diabetic macular edema, uveitic macular edema,central retinal vein occlusion, branch retinal vein occlusion, cornealneovascularization, retinal neovascularization, ocular histoplasmosis,neovascular glaucoma, retinoblastoma, and the like, and combinationsthereof). Additionally, a “subject in need” is also used herein to referto an individual at risk for or diagnosed by a medical professional ashaving ocular neovascularization or a disease or disorder related toocular neovascularization. As such, in some embodiments, the methodsdisclosed herein are directed to a subset of the general population suchthat, in these embodiments, not all of the general population maybenefit from the methods. Based on the foregoing, because some of themethod embodiments of the present disclosure are directed to specificsubsets or subclasses of identified individuals (that is, the subset orsubclass of subjects “in need” of assistance in addressing one or morespecific conditions noted herein), not all individuals will fall withinthe subset or subclass of individuals as described herein. Inparticular, the individual in need is a human. The individual in needcan also be, for example, a research animal such as, for example, anon-human primate, a mouse, a rat, a rabbit, a cow, a pig, and othertypes of research animals known to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure belongs.

Various functions and advantages of these and other embodiments of thepresent disclosure will be more fully understood from the examples shownbelow. The examples are intended to illustrate the benefits of thepresent disclosure, but do not exemplify the full scope of thedisclosure.

EXAMPLES Example 1

In this Example, APX2009 and APX2014 were analyzed for their function onAP-1 DNA binding and cell proliferation and migration.

Materials and Methods

Synthetic Methods. The compounds were synthesized by Cascade CustomChemistry (Eugene, OR) and provided by Apexian Pharmaceuticals. Insummary (FIG. 1A), iodolawsone (2-iodo-3-hydroxy-1,4 naphthoquinone) wasmade available from Cascade Custom Chemistry. HPLC were performed usingan Alltech Alltima column C18 5u, 250×5.6 mm, flow 1 mL/min at 40° C.Elution was with a mobile phase of 15:10:75 water:A1:methanol where A1was made using 700 mL of water, 300 mL methanol and 3 mL trimethylamineto which phosphoric acid was added to bring the pH to 3.4.

(E)-2-((3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylene)pentanoicacid (3). In a 2 L 3-necked flask equipped with a mechanical stirrer anda gas dispersion fritted tube was placed 2-iodo-3-hydroxy-1,4naphthoquinone, (iodolawsone, 1) (18 g, 0.06 mol) and 2-propylacrylicacid 2 (17.1 g, 0.15 mol) in a solution of potassium carbonate (41.4 g,0.3 mol) in water (600 mL). The reaction mixture was stirred and spargedwith argon for 30 minutes. Palladium(II) acetate (0.67 g, 0.003 mol) wasadded and sparging continued for an additional 30 minutes. The resultingmixture was heated in an oil-bath at 100° C. HPLC analysis showed thereaction was complete after 1 hour. The reaction mixture was cooled toroom temperature and the black Pd metal was filtered. The filtrate wasplaced in a 2 L 3-necked flask equipped with a mechanical stirrer,cooled in an ice-methanol bath and acidified with 50% H₃PO₄ (160 mL) topH 2. After stirring for 1 hour, the solid was collected, washed withwater (1 L), a mixture of 20% acetone in water (500 mL), and air driedto give 12.3 g (72%) of (3) as a mustard colored solid. HPLC analysisshowed a purity of 98%. NMR (d₆-DMSO) δ 12.6 (br s, 1H), 11.65 (br s,1H), 8.0 (m, 2H), 7.8 (m, 2H), 7.15 (s, 1H), 2.1 (m, 2H), 1.4 (m, 2H),0.8 (m, 3H).

(E)-2-((3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylene)pentanoylchloride (4). To a suspension of (3) (4.0 g, 0.014 mol) and DMF (0.1 mL)in dichloromethane (75 mL) was added oxalyl chloride (17.5 mL of 2M inCH₂Cl₂, 0.035 mol) over 20 minutes at room temperature. The resultingmixture was stirred at room temperature overnight and then wasconcentrated under reduced pressure to give 4.5 g (100%) (4) as a brownsolid. This solid was used directly in the next step. NMR (CDCl₃) δ7.8-8.2 (m, 2H), 7.7-7.8 (m, 2H), 7.4 (s, 1H), 2.1-2.4 (m, 2H), 1.2-1.7(m, 2H), 0.6-1.0 (m, 3H).

(E)-N,N-diethyl-2-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylene)pentanamide(5a). To a solution of crude (4) (9.7 g, 0.03 mol) in dichloromethane(50 mL) was a solution of diethylamine hydrochloride (4.97 g, 0.045 mol)and diisopropylamine (11.6 g, 0.09 mol) in dichloromethane (50 mL) atroom temperature over 45 minutes. HPLC analysis after 15 minutes showedthe reaction was complete. The reaction mixture was washed with water(100 mL), 1 M HCl (2×100 mL), and brine (100 mL). The organic phase wasdried with 1PS paper and concentrated to a deep red solid. The solid wasflash chromatographed over silica gel (150 g) with anhydrous sodiumsulfate (20 g) on top packed with hexane. The column was eluted with 125mL portions of 15% ethyl acetate in hexane for fractions 1-4, 25% ethylacetate in hexane for fractions 5-8, 35% ethyl acetate in hexane forfractions 9-16, and 50% ethyl acetate in hexane for fractions 17-32. Allfractions were checked by TLC (ethyl acetate:hexane; 1:1) and somefractions by HPLC. The product was eluted in fractions 21 to 30. Theywere combined and concentrated under reduced pressure to give an orangesolid. This solid was suspended over 15% ethyl acetate in hexane (50 mL)and stirred for 15 minutes. The solid was collected and air dried togive 6.7 g (62%) of (5a) as an orange solid. HPLC analysis showed apurity of 99%. NMR (CDCl₃) δ 8.1-8.3 (m, 2H), 7.7-7.8 (m, 2H), 6.1 (s,1H), 3.6 (br d, 4H), 2.2 (t, 2H), 1.45 (m, 2H), 1.25 (br s, (6H), 0.9(t, 3H).

(E)-N-methoxy-2-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylene)pentanamide(5b). To a solution of crude (4), prepared from (3) (20.0 g, 0.7 mol)with DMF (0.5 mL) in DCM (300 mL) and oxalyl chloride (2 M in DCM, 87.5mL, 0.0175 mol), in 100 mL DCM and added to a solution of methoxyaminehydrochloride (7.0 g, 0.084 mol) and DIPEA (27.1 g, 0.21 mol) in DCM(100 mL) under argon and cooled in a room temperature water bath over 1hour. After 30 minutes, HPLC indicated the reaction was complete. Themixture was washed with water (100 mL), 1 M HCl (100 mL), and brine (100mL). The organic phase was dried with 1PS paper and concentrated to anorange oil. The crude oil was chromatographed on silica gel (350 g) withhexanes/EtOAc. The product eluted with 60% EtOAc/hexanes. The purefractions were combined to give 19 g of an oil that solidified. Thesolid was triturated with hexanes (100 mL) and filtered to give 16.6 gof (5b) as a yellow solid (71%) at 98% purity.

(E)-N,N-diethyl-2-((3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylene)pentanamide(6a). To a solution of (5a) (5.0 g, 0.014 mol) in methanol (100 mL) wasadded a solution of sodium methoxide in methanol (4.2 mL of 5 M in MeOH)in one portion sparged with argon. After 30 minutes, HPLC indicated thereaction was complete. The reaction mixture was acidified to pH 3 byusing 3 M HCl (3.5 mL), and then was concentrated under reducedpressure. The resulting residue was dissolved in ethyl acetate (150 mL),washed with water (2×75 mL), and brine (1×100 mL), filtered through 1PSfilter paper and concentrated under reduced pressure to give an oilwhich solidified. This solid was triturated with hexane (50 mL) for 30minutes and the solid was collected and air dried to give 4.8 g (96%) of(6a), APX2009, as a light orange solid. HPLC analysis showed a purity of99%. NMR (CDCl₃) δ 8.15 (m, 2H), 7.75 (m, 2H), 6.2 (s, 1H), 4.1 (s, 3H),3.6 (br d, 4H), 2.2 (t, 2H), 1.4 (m, 4H), 1.25 (br d, 4H), 0.85 (t, 3H).

(E)-N-methoxy-2-((3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylene)pentanamide(6b). To a solution of (5b) (10.0 g, 0.03 mol) in methanol (100 mL) wasadded a solution of sodium methoxide in methanol (9.0 mL of 5M in MeOH)in one portion sparged with argon. After 30 minutes, HPLC indicated thereaction was complete. The mixture was acidified to pH 2-3 with 3 M HCl.The mixture was concentrated under reduced pressure to a residue. Theresidue was dissolved in ethyl acetate (150 mL) and washed with water(100 mL) and brine (100 mL). The organic phase was dried over 1PS paperand concentrated under reduced pressure to an oil that solidified. Thesolid was triturated with hexanes (150 mL) for 30 minutes and filteredto give 8.7 g (83%) of (6b), APX2014, as a yellow solid. HPLC analysisshowed a purity of 99%. NMR (CDCl₃) δ 8.8 (br s, 1H), 8.1 (m, 2H), 7.75(m, 2H), 6.7 (s, 1H), 4.15 (s, 3H), 3.9 (s, 3H), 2.2 (m, 2H), 1.4 (m,2H), 0.85 (t, 3H).

APX3330 was synthesized as described in Luo et al., Antioxid RedoxSignal 10:18531867 (2008).

Electrophoretic mobility shift assays (EMSA). These assays wereperformed as previously described (Luo et al., Antioxid Redox Signal10:18531867 (2008); Kelley et al., Antioxid Redox Signal 14:1387-1401(2011); Su et al., Biochemistry 50:82-92 (2011); Luo et al.,Biochemistry 51:695-705 (2012); Zhang et al., Biochemistry 52:2955-2966(2013)). Briefly, an increasing amount of APX3330, APX2009 or APX2014was pre-incubated with purified Ref-1 protein in EMSA reaction bufferfor 30 minutes. The EMSA assay was performed using the AP-1 target DNAsequence and AP-1 protein.

Cells. Primary human retinal microvascular endothelial cells (HRECs)were obtained from Cell Systems, Inc. (Kirkland, WA), while the Rf/6amacacque choroidal endothelial cell line was obtained from ATCC(Manassas, VA). Cells were maintained as described (Basavarajappa etal., EMBO Mol Med 9:786-801 (2017)), re-ordered at least annually, andregularly assessed for mycoplasma contamination.

In vitro cell proliferation assay. Endothelial cell proliferation wasmeasured as described previously (Basavarajappa et al., PLoS One9:e95694 (2014); Basavarajappa et al., EMBO Mol Med 9:786-801 (2017)).Briefly, 2.5×10³ cells were seeded in 100 μL of growth medium and platedin each well of 96-well clear-bottom black plates and incubated for 24hours. APX2009, APX2014, or DMSO vehicle (DMSO final concentration=1%)was added, and the plates were incubated for 24-48 hours in 100 μLcomplete medium at 37° C. and 5% CO₂. AlamarBlue reagent (11.1 μL) wasadded to each well of the plate and 4 hours later fluorescence readingswere taken at excitation and emission wavelengths of 560 nm and 590 nm,respectively, using a Synergy H1 plate reader (BioTek, Winooski, VT).GI50 was calculated using GraphPad Prism v. 7.0.

EdU incorporation, Ki-67 Staining and TUNEL. These assays were carriedout as described previously (Basavarajappa et al., PLoS One 9:e95694(2014); Basavarajappa et al., EMBO Mol Med 9:786-801 (2017)) with theexception of using chamber slides, not coverslips. Briefly, cells(30,000 per well) were seeded on 8-well chamber slides coated withattachment factors and allowed to attach overnight. Cells were treatedwith the indicated compound concentrations for 17 hours (overnight). Toassay proliferation, cells were incubated with EdU in complete media for8 hours at 37° C. Cells were then fixed in 4% paraformaldehyde for 20minutes and permeabilized using 0.25% Triton X-100 prepared in PBS.Cells were incubated with a rabbit-specific monoclonal antibody againstKi-67 (D3B5) (#9129; Cell Signaling, Danvers, MA) (1:400) overnight at4° C. Secondary antibody was Alexa Fluor goat anti-rabbit 488 (A11034;Invitrogen, Carlsbad, CA) with DAPI counter-stain for nuclear staining.Proliferating cells that incorporated EdU were detected using theClick-iT EdU Imaging kit (Invitrogen, Carlsbad, CA). Alternatively,apoptotic cells were visualized using the Click-iT TUNEL assay kit(Invitrogen, Carlsbad, CA) as per the manufacturer's instructions, withHoechst 33342 counter-stain for nuclear staining, and a 17-hourtreatment with 1 μM staurosporine as positive control. The cells wereimaged using a Zeiss Axiolmager D2 microscope or an LSM 700 confocalmicroscope and the percentage of positive cells was counted on threelow-power (for TUNEL) or high-power (for Ki-67 and EdU) fields per wellusing ImageJ software.

Cell cycle analysis. HRECs (2×10⁶) were grown in EGM-2 medium. Cellswere serum starved in EBM-2 medium overnight, then treated with theindicated concentrations of APX2009 or APX2014 along with DMSO controlfor 24 hours in complete medium. Cells were washed twice in ice-cold PBSfollowed by fixation in 66% ethanol solution overnight at 4° C. Fixedcells were again washed twice in ice-cold PBS and the pellets wereresuspended in propidium iodide staining solution for 30 minutes at 37°C. (20 μg/mL propidium iodide prepared in 1×PBS containing 0.1% TritonX-100 and 100 μg/mL RNase A). After incubation cells were analyzed usingflow cytometry (FACSCalibur, BD Biosciences, San Jose, CA). Pulse shapeanalysis was used to exclude doublets and debris. The single cellpopulation was then assessed by the FL2 area histogram plot using ModFitsoftware (v. 5.0) and cell cycle profiles were generated.

In vitro cell migration assay. Endothelial cell migration was monitoredas described before (Basavarajappa et al., PLoS One 9:e95694 (2014);Basavarajappa et al., EMBO Mol Med 9:786-801 (2017)). Briefly, HRECs andRf/6a were grown until confluency in 12-well plates. Using a sterile10-μL micropipette tip, a scratch wound was made across the center ofeach well and fresh complete media containing DMSO or differentconcentrations of APX2009 or APX2014 compounds were added to the wells(DMSO final concentration=1%). Wells were imaged via digital brightfieldmicroscopy at different time points, and the number of migrated cellsinto the scratched area was manually counted.

In vitro Matrigel tube formation assay. The ability of HRECs and Rf/6acells to form tubes in vitro was monitored as described before(Basavarajappa et al., PLoS One 9:e95694 (2014); Basavarajappa et al.,EMBO Mol Med 9:786-801 (2017)). Briefly, cells were treated with theindicated concentrations of APX2009 or APX2014 compounds or DMSO for 48hours and then 1.5×10⁴ cells in 100 μL of growth medium containing DMSOor APX compounds were added to each well of a 96-well plate that waspre-coated with 50 μL of Matrigel basement membrane (DMSO finalconcentration=1%). Digital photographs of each well at different timepoints were taken to measure the in vitro tube formation using theAngiogenesis Analyzer plugin in ImageJ software (v.1.48;http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzer-for-ImageJ.html).

NF-κB p65 nuclear translocation assay. The NF-κB nuclear translocationassay was performed by seeding 30,000 HRECs/well on an 8-well chamberslide coated with attachment factors. The cells were grown in EGM-2medium overnight before treating with indicated concentrations ofAPX2009 and APX2014, or 10 μM BAY 11-7082 (Sigma, St. Louis, MO) as apositive control NF-κB inhibitor. After 17 hours incubation, the mediawas replaced with EBM-2 (minimal medium) with indicated concentrationsof compound or DMSO for 1 hour. The cells were then stimulated with 10ng/ml TNF-α in EBM-2 for 20 minutes at 37° C. to activate NF-κB. Cellswere then fixed in 4% paraformaldehyde and permeabilized using 0.5%Triton X-100 solution prepared in PBS. The cells were incubated with amonoclonal antibody against NF-κB p65 (sc-8008; Santa Cruz, Santa Cruz,CA) (1:50) overnight at 4° C., followed by Alexafluor 555 goatanti-mouse secondary antibody (1:2000) for one hour. The cells werecounter-stained with Hoechst 33342 for nuclear staining and then mountedusing Everbrite hardset mounting medium. The cells were imaged using aZeiss AxioImager D2 microscope.

qRT-PCR. The assay was performed as described previously (Basavarajappaet al., PLoS One 9:e95694 (2014); Basavarajappa et al., EMBO Mol Med9:786-801 (2017)). RNA was extracted from cells treated as indicatedusing Trizol (Invitrogen). cDNA was synthesized from 1 μg RNA usingrandom primers and iScript reverse transcriptase (Bio-Rad, Hercules,CA). qPCR was performed in 10 μL volumes in a 384-well plate, with FastAdvanced Master Mix and TaqMan probes on a ViiA7 thermal cycler (AppliedBiosystems, Foster City, CA). Primer/probesets used were as follows:VEGFA (Hs00900055_m1), VCAM1 (Hs01003372_m1), and CCL20 (Hs01011368_m1),and housekeeping controls HPRT (Hs02800695_m1) and TBP (Hs00427620_m1).The data were analyzed using the AACt method. The expression levels ofgenes were normalized to the two housekeeping genes and calibrated tothe DMSO treated sample.

Animals. All animal experiments were approved by the Indiana UniversitySchool of Medicine Institutional Animal Care and Use Committee andfollowed the guidelines of the Association for Research in Vision andOphthalmology Statement for the Use of Animals in Ophthalmic and VisualResearch. Wild-type female C57BL/6 mice, 6-8 weeks of age, werepurchased from Envigo (Indianapolis, IN; for choroidal sproutingexperiments) or Jackson Laboratory (Bar Harbor, ME; for L-CNV) andhoused under standard conditions (Wenzel et al., Mol Vis 21:515-522(2015)). Mice were anesthetized for all procedures by intraperitonealinjections of 90 mg/kg ketamine hydrochloride and 5 mg/kg xylazine, withintraperitoneal atipamezole reversal (1 mg/kg). Treatments were randomlyassigned by cage.

Choroidal sprouting assay. Ex vivo Choroidal sprouting was assessed asdescribed previously (Sulaiman et al., Sci Rep 6:25509 (2016);Basavarajappa et al., EMBO Mol Med 9:786-801 (2017)). Briefly,choroid-sclera was dissected from 7 to 8 week old mouse eyes and pieceswere embedded in Matrigel (growth factor reduced) and grown in EGM-2medium containing antibiotics for 72 hours to allow sprouting toinitiate. The indicated concentrations of APX2009 and APX2014 compounds(in DMSO, final DMSO concentration 0.5 and 0.2%, respectively) wereadded and growth allowed to proceed for 48 hours. Images were taken andgrowth was quantified by measuring the distance from the edge of thechoroidal piece to the growth front in four directions per sample usingImageJ software.

Laser-induced choroidal neovascularization. L-CNV was induced asdescribed previously (Sulaiman et al., J Ocul Pharmacol Ther 31:447-454(2015); Sulaiman et al., Sci Rep 6:25509 (2016); Basavarajappa et al.,EMBO Mol Med 9:786-801 (2017)). Studies were powered to have an 80%chance of detecting effect size differences of 50%, assuming 30%variability, α=0.05. Briefly, pupils of anesthetized mice were dilatedwith 1% tropicamide (Alcon Laboratories Inc., Fort Worth, TX) andlubricated with hypromellose ophthalmic demulcent solution (Gonak)(Akorn, Lake Forest, IL). A coverslip was used to allow viewing of theposterior pole of the eye. Three burns of a 532 nm ophthalmic argongreen laser coupled with a slit lamp (50 μm spot size, 50 ms duration,and 250 mW pulses) were delivered to each 3, 9, and 12 o'clock position,two-disc diameters from optic disc. The bubbling or pop sensed afterlaser photocoagulation was considered as the successful rupture ofBruch's membrane. Lesions in which bubbles were not observed wereexcluded from this Example. To assess the antiangiogenic activity ofAPX3330, the mice were i.p. injected with compound (50 mg/kg bodyweight), twice daily, five days on/two days off, as used previously invivo (Fishel et al., Mol Cancer Ther 10:1698-1708 (2011); Lou et al.,Oncol Lett 7:1078-1082 (2014); Biswas et al., Am J Physiol Cell Physiol309:C296-307 (2015)). Vehicle was 42% Cremophor: 2% ethanol in PBS. ForAPX2009, doses were 12.5 mg/kg or 25 mg/kg body weight, twice dailyuntil 14 days of laser treatment unless otherwise indicated. Vehicle waspropylene glycol, Kolliphor HS15, Tween 80 (PKT) (McIlwain et al.,Oncotarget doi.org/10.18632/oncotarget.23493. (2017)). Mice were weigheddaily.

In vivo imaging. Optical coherence tomography (OCT) was performed inL-CNV mice as described previously (Sulaiman et al., Sci Rep 6:25509(2016)), at the indicated times using the Micron III intraocular imagingsystem (Phoenix Research Labs, Pleasanton, CA). Briefly, before theprocedure, eyes of anesthetized mice were dilated with 1% tropicamidesolution (Alcon, Fort Worth, TX) and lubricated with hypromelloseophthalmic demulcent solution (Gonak) (Akorn, Lake Forest, IL, USA).Mice were then placed on a custom heated stage that moves freely toposition the mouse eye for imaging. Several horizontal and vertical OCTimages were taken per lesion. Fluorescein angiography was performed 14days post laser by intraperitoneal injection of 50 μL of 25% fluoresceinsodium (Fisher Scientific, Pittsburgh, PA). Fundus images were takenusing the Micron III system and Streampix software.

Choroidal flatmount immunofluorescence. Mouse eyes were harvested 14days after L-CNV induction. The eyes were enucleated and fixed in 4%paraformaldehyde/PBS overnight. The anterior segment, lens, and retinawere removed, and the posterior eye cups were prepared for choroidalflat mounts. The posterior eye cups were washed with PBS andpermeabilized in blocking buffer containing 0.3% Triton X-100, 5% bovineserum albumin (BSA) in PBS for two hours at 4° C. After blocking, theeye cups were double stained for vasculature with rhodamine-labeledRicinus communis agglutinin I (Vector Labs, Burlingame, CA) and AlexaFluor™ 488 conjugated-Isolectin B4 from Griffonia simplicifolia (GS-IB4)(Molecular Probes, Thermo Fisher Scientific) at 1:250 concentration inbuffer containing 0.3% Triton X-100, 0.5% BSA in PBS for 16-20 hours at4° C. After antibody incubation, whole mounts were washed three timeswith PBS for 15 minutes each step at 4° C. with 0.1% Triton X-100. Afterwashing, choroidal flatmounts were mounted in aqueous mounting medium(VectaShield; Vector Laboratories, Inc.) and cover-slipped forobservation by confocal Z-stack imaging (LSM 700, Zeiss, Thornwood, NY)to estimate lesion volume. The sum of the stained area in each opticalsection, multiplied by the distance between sections (3 μm), gave theCNV lesion volume and lesion volume was quantified using ImageJsoftware. Lesions were only included for analysis if they met qualitycontrol standards as published (Poor et al., Invest Ophthalmol Vis Sci55:6525-6534 (2014)). All lesions in an eye were averaged to represent asingle n.

Statistical analyses. Statistical analyses were performed with GraphPadPrism 7 software. One-way ANOVA was used with Dunnett's post hoc testfor migration, tube formation, and choroidal sprouting experiments.One-way ANOVA was used with Tukey's post hoc test for APX2009 in vivoexperiments. Unpaired Student's t-test was used for the APX3330 in vivoexperiment. Two-sided p values<0.05 were considered statisticallysignificant.

Results

Ref-1 inhibitors APX2009 and APX2014 were more potent than APX3330.APX2009 (6a) and APX2014 (6b) (FIG. 1A) was synthesized and demonstratedthat both compounds had enhanced inhibition of Ref-1-inducedtranscription factor binding to DNA compared to APX3330 (7) (FIG. 1 i ),while having substantially different physiochemical properties. The newcompounds have lower molecular weights, and lack the carboxylate groupand long alkyl chain of APX3330. The new compounds also havesignificantly reduced lipophilicity as determined by computer basedcalculation of their c log P values, APX3330=4.5, APX2009=2.7, andAPX2014=1.9.

APX2009 and APX2014 blocked endothelial cell proliferation. Endothelialcell proliferation with increased survival supports the cells that makeup new blood vessels, leading to angiogenesis. Proliferation assays werecarried out to measure angiogenic or antiangiogenic activity. As aninitial test of the antiangiogenic potential of our these two new Ref-1inhibitors, their ability to inhibit the proliferation of HRECs andRf/6a choroidal endothelial cells (FIGS. 2A-2D) was assessed. Bothcompounds dose-dependently blocked proliferation of both cell types inan alamarBlue assay, with APX2014 more potent than APX2009. PrimaryHRECs were more sensitive to both compounds than the Rf/6a choroidalcell line, as seen for other antiangiogenic compounds.

APX2009 and APX2014 blocked S phase without inducing apoptosis. Theactivity of the compounds were assessed in more detail in HRECs. Bothcompounds reduced the number of cells going through S phase as evidencedby reduced Ki-67 staining and reduced EdU incorporation (FIGS. 3A-3D;FIGS. 4A & 5 ). This was also evident as a modest increase in cells inG0/G1 phase at high doses of compound, with a concomitant decrease inG2/M phase cells (FIGS. 4B & 4C). However, neither compound inducedapoptosis at anti-proliferative doses as assessed by TUNEL (FIGS. 6A &6B).

APX2009 and APX2014 blocked endothelial cell migration.Neovascularization involves an array of coordinated events, includingextracellular matrix degradation, cell migration, cell proliferation,and morphogenesis of endothelial cells. To know the effect of APX2009and APX2014 compounds on endothelial cell migration, a scratch-woundassay was performed. (FIGS. 7A-7D; FIGS. 8A-8D). Both compounds againwere dose-dependently effective here, without causing obviouscytotoxicity over the short time course of these assays.

APX2009 and APX2014 blocked endothelial cell tube formation. Endothelialcells organize and form capillary-like structures upon plating on anextracellular matrix such as Matrigel. The organization of endothelialcells into a three-dimensional network of tubes is essential forangiogenesis. As such, the Matrigel tube formation assay is a good invitro predictor of angiogenic potential in vivo. In this assay, bothAPX2009 and APX2014 inhibited tubule formation markedly, atconcentrations lower than those required for inhibiting migration alone,strongly indicative of antiangiogenic activity (FIGS. 9A-9D; FIGS.10A-10D).

APX2009 and APX2014 inhibited NF-κB activity. Since Ref-1 inhibition haspreviously been associated with reduction in NF-κB activity (Shah etal., 2017), the activity of this pathway was assessed in response to thecompounds in HRECs, to determine if APX2009 and APX2014 were actingthrough the expected mechanisms. First, the translocation of the p65subunit of NF-κB into the nucleus was assessed in response to TNF-α, akey indication of pathway activity. Translocation of p65 wasdose-dependently attenuated in APX2009 and APX2014-treated HRECs (FIG.11A). Moreover, production of the mRNA of VEGFA, VCAM1, and CCL20, allof which are downstream of NF-κB, was decreased 3- to 10-fold by thesecompounds (FIGS. 11B, 11C & 11D).

APX2009 and APX2014 blocked angiogenesis ex vivo. As a further test ofactivity, a choroidal sprouting assay using murine choroidal explantswas used to test the efficacy of the APX compounds in a complexmicrovascular bed in tissues (FIGS. 12A-12D). In this assay, choroidalcells grow out of the choroidal tissue piece into a surrounding Matrigelmatrix. Both compounds significantly reduced sprouting, with APX2014remaining more potent. At 10 μM, APX2009 reduced sprouting by ˜70%compared to control (FIGS. 12A & 12B), while at 1 μM (the highestconcentration tested), APX2014 reduced sprouting by ˜60% compared tocontrol (FIGS. 12C & 12D).

Systemic Ref-1 inhibition with parent compound APX3330 can preventL-CNV. Previous efforts to attenuate ocular neovascularization by Ref-1inhibition using APX3330 relied on intravitreal delivery of compound.Although this is the delivery route of the standard-of-care anti-VEGFbiologics and ensures that the drug gets to the right place in humans,it is labor-intensive, causes patient discomfort, and incurs a risk ofpotentially vision-threatening endophthalmitis. Thus, it was explored ifsystemic (intraperitoneal) Ref-1 inhibition could offer an alternativeroute to therapy of L-CNV. As a proof-of-concept, i.p. injections of thefirst-generation Ref-1 inhibitor APX3330 (7) delivered 50 mg/kg twicedaily, 5 days on/two days off, for two weeks was employed. This dosingregimen was chosen as it was previously successful and non-toxic forpreclinical tumor studies. Animals treated with APX3330 displayedsignificantly reduced L-CNV volume (FIGS. 13A-13C).

Systemic administration of more potent derivative APX2009 reduced L-CNVsignificantly. Given that APX3330 was an effective systemic agent forL-CNV, the effects of the new second-generation Ref-1 inhibitors wereanalyzed.

APX2009 was chosen for this experiment as it had previously been safelydosed in animals. Two dosage regimens previously employed, 12.5 or 25mg/kg, twice daily for two weeks were used. The lower dose did notreduce L-CNV, but the 25 mg/kg dose had a marked effect (FIGS. 14A-14D).This was qualitatively evident by OCT imaging on Day 7, and even moresubstantial on Day 14 (FIG. 14A). In addition, qualitatively lessfluorescein leakage was seen in lesions by fluorescein angiography atDay 14 (FIG. 14B). Finally, L-CNV lesion volume assessed by ex vivostaining with agglutinin (FIG. 14C) and isolectin B4 (FIGS. 15A-15C),was reduced by 25 mg/kg APX2009 approximately four-fold compared tovehicle (FIG. 14D).

The observed effects are likely attributable to redox signalinginhibition, rather than DNA repair inhibition, as the compounds arespecific for redox signaling inhibition. The molecularly distinctfunctional portions of Ref-1, redox and DNA repair, are completelyindependent. For example, mutations of the cysteine at position 65(C65A) of APE1/Ref-1 abrogate the redox function, but do not affect DNArepair function, and vice versa. Moreover, Ref-1 inhibitors such asAPX3330 do not inhibit APE1 activity. In fact, APX3330 and APX2009 canenhance APE1 repair activity in neurons, potentially contributing to aneuroprotective effect of these agents, which could offer an addedbenefit in the context of photoreceptor cell death in neovascular eyediseases.

Given their anti-Ref-1 redox signaling activity, APX2009 and APX2014likely exert their antiangiogenic effects by blocking the activation oftranscription factors induced by Ref-1. Likely candidates include NF-κBand HIF-1α, both of which can regulate VEGF. In retinal pigmentepithelial cells, APX3330 reduced both NF-κB and HIF-1α activity, with aconcomitant reduction in VEGF expression. Additionally, APX3330treatment of stroke in type one diabetes mellitus rats significantlydecreased total vessel density and VEGF expression. The exacttranscription factors modulated by Ref-1 inhibition in the context ofocular neovascularization remain to be determined, however.

There was not observed obvious intraocular or systemic toxicity of thetwo compounds tested in vivo (APX3330 and APX2009), nor was substantialcell death seen in migration, tube formation, and choroidal sproutingassays. These findings are consistent with the excellent safety profilefor APX3330 in humans. Nonetheless, ocular toxicity of the new compoundsand intraocular pharmacokinetics remain to be thoroughly examined.

A well-tolerated, systemic drug therapy has significant potential fortreatment of neovascular eye diseases. The existing approved drugs areall biologics requiring intravitreal injection in the context of anophthalmologist's clinic. An orally bioavailable drug (as with APX3330)could be administered at home, potentially as a once daily pill. Thetradeoff for such a therapy would be much more frequent dosing than thatrequired for intravitreal injections (monthly or less), and moresubstantial systemic exposure than that seen with intravitrealtherapies. But given the strong safety profile of Ref-1 inhibitors, thismight be manageable. Moreover, patient and healthcare system costs mightbe significantly lower with such a therapy, as office visits andinjection procedures could be reduced.

In summary, it has now been shown for the first time that systemicadministration of Ref-1 inhibitors (APX2009 and APX2014) can attenuateL-CNV. As L-CNV is a widely-used model of the choroidalneovascularization that underlies wet AMD, suggesting that Ref-1inhibition could find therapeutic utility for this indication. The invitro data suggest that Ref-1 inhibition also effectively blocksangiogenesis involving retinal endothelial cells. Thus, these inhibitorsmay also be useful for retinal neovascular diseases like ROP and PDR.

Example 2

In this Example, the effects of Ref-1 knockdown on NF-κBsignaling-associated genes was analyzed.

Human retinal endothelial cells (HRECs) (Cell Systems, Inc. Kirkland,WA) were plated in 6-well plates and treated with 0.1 μM APX2009, 1 μMAPX2009 or DMSO for 6 hours and 24 hours. Cells were washed once aftertreatment with PBS, collected and frozen. This process was repeated tocollect treated cells in 4 different passages. RNA was extracted in 300μL Trizol (Life Technologies, Carlsbad, CA) and flash frozen at −80° C.The SMARTer system (Clontech, Mountain View, CA) was used to generatecDNA from cells. The dscDNA quantity and quality was assessed using anAgilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) withthe High Sensitivity DNA Chip. A total of 48 SCR and 48 siAPE1 cellswere chosen for sequencing. The IUSM Genomics Facility preparedlibraries using a Nextera kit (Illumina, San Diego, CA). DNAs weresequenced using the Illumina HiSeq 4000.

Ref-1 activates NF-κB and HIF-1α signaling: Redox-dependentstabilization of the HIF-1α protein is required for activation of HIF-1,and redox signaling through Ref-1 regulates the DNA-binding activity ofHIF-1. Hypoxia-driven gene expression is not solely through HIF; otherTFs that respond to hypoxia include NF-κB, AP-1, and others. Molecularchanges induced by hypoxia can impact upon angiogenesis, both in the eyeand in cancer. Novel compounds APX2009 and APX2014 inhibit NF-κBactivation and reduce target gene expression in HRECs. This wasconfirmed in this RNA-Seq experiment demonstrating that APX2009 blockedHIF regulated genes in a concentration dependent manner (FIG. 16 ).

Example 3

In this Example, the role of Ref-1 on ocular angiogenesis is analyzed.

The Protein Atlas was mined for expression data on Ref-1. In addition,immunohistochemistry was performed for Ref-1 in de-identified postmortemeye tissue from a wet AMD patient and an age-matched control. Tissuesections were deparaffinized. DAB was used for detection andcounterstained with DAPI. Images of retina and choroid were taken on anEVOS fl digital microscope.

Ref-1 is highly expressed in developing murine retinas, as well asretinal pigment epithelium (RPE) cells, retinal pericytes, choroidalendothelial cells (CECs) and retinal endothelial cells (RECs). At theRNA level, it is expressed higher in retina than in all but a third of36 other tissue types(https://www.proteinatlas.org/ENSG00000100823-APEX1/tissue). Preliminaryevidence also suggests that it is upregulated in the retina and choroidof human wet AMD patient eyes compared with age-matched controls (FIG.17 ), suggesting disease relevance.

What is claimed is:
 1. A method of inhibiting ocular neovascularization in a subject in need thereof, the method comprising administering to the subject an effective amount of an apurinic/apyrimidinic endonuclease 1 redox factor 1 (APE1/Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.
 2. The method as set forth in claim 1, wherein the APE1/Ref-1 inhibitor has the formula:

wherein R₁ is selected from the group consisting of alkyl, alkoxy, hydroxyl, and hydrogen; R₃ and R₆ are independently selected from the group consisting of a substituted or unsubstituted alkoxy, a substituted or unsubstituted aryl and an oxo; R₄ and R₅ are independently selected from the group consisting of an alkoxy and aryl, or both R₄ and R₅ taken together form a substituted or unsubstituted napthoquinone; X is selected from the group consisting of CH═CR₂ and NCH, wherein R₂ is selected from the group consisting of C₁-C₁₀ alkyl and CF₃CH₂CH₂; and Y is selected from the group consisting of N(Rz)R2 or NR{circumflex over ( )}OR{circumflex over ( )}, wherein each Rz is independently selected from the group consisting of C₁-C₆ alkyl, heteroalkyl, cycloalkyl and cycloheteroalkyl, straight or branched chain or optionally substituted, or both Rz and R2 taken together with the attached nitrogen form an optionally substituted heterocycle; where each R{circumflex over ( )} is independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cyclohexyl, and cycloheteroalkyl, each of which is optionally substituted, or both R{circumflex over ( )} are taken together with the attached nitrogen and oxygen to form an optionally substituted heterocycle.
 3. The method as set forth in claim 1 further comprising administering at least one additional therapeutic agent to the subject.
 4. The method as set forth in claim 3, wherein the additional therapeutic agent is selected from the group consisting of an anti-VEGF treatment, vitamins, minerals and combinations thereof.
 5. The method as set forth in claim 4, wherein the anti-VEGF treatment is selected from the group consisting of ranibizumab, bevacizumab, aflibercept, and combinations thereof.
 6. The method as set forth in claim 1, wherein the subject has a disease selected from the group consisting of retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), diabetic retinopathy, wet age-related macular degeneration (AMD), pathological myopia, hypertensive retinopathy, occlusive vasculitis, polypoidal choroidal vasculopathy, diabetic macular edema, uveitic macular edema, central retinal vein occlusion, branch retinal vein occlusion, corneal neovascularization, retinal neovascularization, ocular histoplasmosis, neovascular glaucoma, retinoblastoma, and combinations thereof.
 7. A method of treating retinopathy of prematurity (ROP) in a subject in need thereof, the method comprising administering to the subject an effective amount of an apurinic/apyrimidinic endonuclease 1 redox factor 1 (APE1/Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.
 8. The method as set forth in claim 7, wherein the APE1/Ref-1 inhibitor has the formula:

wherein R₁ is selected from the group consisting of alkyl, alkoxy, hydroxyl, and hydrogen; R₃ and R₆ are independently selected from the group consisting of a substituted or unsubstituted alkoxy, a substituted or unsubstituted aryl and an oxo; R₄ and R₅ are independently selected from the group consisting of an alkoxy and aryl, or both R₄ and R₅ taken together form a substituted or unsubstituted napthoquinone; X is selected from the group consisting of CH═CR₂ and NCH, wherein R₂ is selected from the group consisting of C₁-C₁₀ alkyl and CF₃CH₂CH₂; and Y is selected from the group consisting of N(Rz)R2 or NR{circumflex over ( )}OR{circumflex over ( )}, wherein each Rz is independently selected from the group consisting of C₁-C₆ alkyl, heteroalkyl, cycloalkyl and cycloheteroalkyl, straight or branched chain or optionally substituted, or both Rz and R2 taken together with the attached nitrogen form an optionally substituted heterocycle; where each R{circumflex over ( )} is independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cyclohexyl, and cycloheteroalkyl, each of which is optionally substituted, or both R{circumflex over ( )} are taken together with the attached nitrogen and oxygen to form an optionally substituted heterocycle
 9. The method as set forth in claim 7 further comprising administering at least one additional therapeutic agent to the subject.
 10. The method as set forth in claim 9, wherein the additional therapeutic agent is selected from the group consisting of an anti-VEGF treatment, vitamins, minerals and combinations thereof.
 11. The method as set forth in claim 10, wherein the anti-VEGF treatment is selected from the group consisting of ranibizumab, bevacizumab, aflibercept, and combinations thereof.
 12. A method of treating wet age-related macular degeneration (AMD) in a subject in need thereof, the method comprising administering to the subject an effective amount of an apurinic/apyrimidinic endonuclease 1 redox factor 1 (APE1/Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.
 13. The method as set forth in claim 12, wherein the APE1/Ref-1 inhibitor has the formula:

wherein R₁ is selected from the group consisting of alkyl, alkoxy, hydroxyl, and hydrogen; R₃ and R₆ are independently selected from the group consisting of a substituted or unsubstituted alkoxy, a substituted or unsubstituted aryl and an oxo; R₄ and R₅ are independently selected from the group consisting of an alkoxy and aryl, or both R₄ and R₅ taken together form a substituted or unsubstituted napthoquinone; X is selected from the group consisting of CH═CR₂ and NCH, wherein R₂ is selected from the group consisting of C₁-C₁₀ alkyl and CF₃CH₂CH₂; and Y is selected from the group consisting of N(Rz)R2 or NR{circumflex over ( )}OR{circumflex over ( )}, wherein each Rz is independently selected from the group consisting of C₁-C₆ alkyl, heteroalkyl, cycloalkyl and cycloheteroalkyl, straight or branched chain or optionally substituted, or both Rz and R2 taken together with the attached nitrogen form an optionally substituted heterocycle; where each R{circumflex over ( )} is independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cyclohexyl, and cycloheteroalkyl, each of which is optionally substituted, or both R{circumflex over ( )} are taken together with the attached nitrogen and oxygen to form an optionally substituted heterocycle
 14. The method as set forth in claim 12 further comprising administering at least one additional therapeutic agent to the subject.
 15. The method as set forth in claim 14, wherein the additional therapeutic agent is selected from the group consisting of an anti-VEGF treatment, vitamins, minerals and combinations thereof.
 16. The method as set forth in claim 15, wherein the anti-VEGF treatment is selected from the group consisting of ranibizumab, bevacizumab, aflibercept, and combinations thereof. 